Summary. Introduction

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1 OsteoArthritis and Cartilage (2001) 9, OsteoArthritis Research Society International /01/ $35.00/0 doi: /joca , available online at on Early changes in lapine menisci during osteoarthritis development: Part I: Cellular and matrix alterations M.-P. Hellio Le Graverand*, E. Vignon, I. G. Otterness and D. A. Hart* *McCaig Center for Joint Injury and Arthritis Research, Faculty of Medicine, University of Calgary, Alberta, Canada T2N 4N1 Claude Bernard University, Faculty of Medicine, Centre Hospitalier Lyon-Sud, Lyon, France Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340, U.S.A. Summary Objective: Osteoarthritis (OA) is the most common form of arthritis and patients with meniscal and ligament injuries of the knee are at high risk to develop the disease. The purpose of this study was to evaluate changes occurring in both medial and lateral menisci from the knees of anterior cruciate ligament (ACL) transected rabbits during the early stages of OA development. Design: Meniscal tissues from control and experimental rabbits were processed for histology and immunohistochemistry for assessment of matrix organization and composition. Results: At 3 and 8 weeks following ACL transection, histological examination demonstrated extensive extracellular matrix deterioration. Altered cell distribution, areas depleted of cells, and areas of cell clusters were found within the medial but not in the lateral meniscus. Immunohistochemistry of both medial and lateral menisci demonstrated significant changes in collagen distribution. Type I and III collagen staining was increased in both medial and lateral menisci. In contrast, type II collagen staining was overtly increased only in the medial meniscus. Conclusion: These results demonstrate that after ACL transection, extracellular matrix deposition as well as altered matrix organization and altered cell distribution occur early in the medial meniscus OsteoArthritis Research Society International Key words: Medial and lateral menisci, Biochemistry of menisci, Experimental osteoarthritis. Introduction Osteoarthritis (OA) is a progressive destructive joint disease characterized by erosive deterioration of joint tissues. As clinical samples are usually obtained from advanced stages of OA, an animal model in which OA is experimentally induced and where the onset time is known offers the opportunity to analyze tissue during the early phases of disease development. One of the most widely used experimental models of OA involves the transection of the ACL. While the ACL transection model of OA was initially developed 1 and extensively characterized 2 5 in the dog, more recently, this surgical procedure has also been used in rabbits Characterization of gross morphological, histological, biochemical and biomechanical changes demonstrated characteristic osteoarthritic lesions of the articular cartilage surfaces in shorter time periods in the rabbit compared with the canine model The changes in various tissues of the knee joint, Received 21 July 1999; accepted 7 April Grant supporters: Medical Research Council of Canada, The Arthritis Society and La Société Française de Rhumatologie. Address correspondence to: Dr David A. Hart, Calgary Foundation-Grace Glaum Professor in Arthritis Research, McCaig Centre for Joint Injury and Arthritis Research, University of Calgary HSC, 3330 Hospital Drive N.W., Calgary, Alberta Canada T2N 4N1. Tel: ; Fax: ; hartd@acs.ucalgary.ca in particular synovium and articular cartilage following ACL transection, have been extensively studied. 11 However, considerably less is known regarding the effect of ACL transection on the knee joint menisci. Adams et al. were the first to describe severe gross morphological changes of the menisci following ACL transection in dogs, including fibrillations and tears of the meniscal tissue. 12 They also reported an early reduction in the meniscal glycosaminoglycan (GAG) content of menisci followed by a return to normal GAG content after 3 18 months postinjury. 12 Subsequently, Sandy et al. demonstrated increased GAG content and increased proteoglycan (PG) synthesis following ACL transection in dogs, in both the medial and lateral meniscus, and that the medial meniscus was more stimulated than the lateral. 13 However, no further investigations into the biochemistry or biomechanical behavior of the menisci following ACL transection have been reported. The menisci are wedge-shaped semi-lunar fibrocartilaginous structures that correct the incongruence of the femoral and tibial articular surfaces. 14 Menisci provide important biomechanical functions to the knee joint such as load bearing, load distribution, shock absorption and joint stability. 15 Because of their functional importance, meniscal repair is usually prefered to the traditional meniscectomy following meniscal injury, in an attempt to preserve the structural integrity of the knee joint and minimize subsequent joint degeneration. Meniscal tissue is composed 56

2 Osteoarthritis and Cartilage Vol. 9 No mainly of type I collagen, while collagen types II, III, V and VI are minor constituents. 14,16,17 In addition, menisci contain considerably less proteoglycan (PG) (<1%) than does hyaline cartilage (7%). 15 Nevertheless, both large chondroitin sulfate and small dermatan sulfate proteoglycans have been shown to be present in meniscal tissue Clinically, the importance of concomitant meniscal and articular cartilage lesions in the femorotibial joint is well known. In a recent retrospective study of 1740 knee joints, the frequency of the association of longitudinal, buckethandle and complex meniscal tears with articular cartilage damage has been described. 22 This study also demonstrated that degeneration of the menisci was significantly correlated with the severity of articular cartilage destruction in both the medial and lateral compartments (P<0.01). 22 Recently, a high incidence of extensive longitudinal tears of the medial meniscus with increased cartilage damage in ACL-deficient knees have also been reported. 23 Based on such clinical evidence, we hypothesized that following ACL transection there would initially be extensive alterations in the medial meniscus, compared to the lateral meniscus, and such changes would precede overt alterations in the cartilage. To address this hypothesis, we studied chronologically the changes in the meniscal tissue during the early stages of OA in the rabbit using histology and immunohistochemistry. Significant reproducible modifications of meniscal structure and organization that were meniscus-specific and time-dependent were demonstrated. Furthermore, it was possible to demonstrate that changes in collagen extracellular matrix deposition occur early following ACL transection. Materials and methods EXPERIMENTAL OA Skeletally mature (12 months of age) female New Zealand white rabbits (Reimans Fur Ranch, St Agatha, ON) were used in the present study. All rabbits had surgery under sterile conditions using a general inhalational anesthetic (1% halothane and oxygen at 1 l/min). Longitudinal skin and fascial incisions were made over the lateral side of the right knee. An anterolateral capsulotomy between the tendon of extensor digitorum longus and the patellar tendon allowed exposure of the ACL. The ACL was then probed with a blunt hook and transected with a no. 15 blade. The joint capsule was then closed with 6-0 nylon suture. Skin incisions were closed with interrupted 4-0 nylon sutures and a dressing of gentian violet was applied to the wound. After surgery, animals were allowed unrestricted activity in cages until sacrifice at 3 and 8 weeks. Age-matched control animals were also sacrificed. All animals received similar diets post-operatively and water ad libitum. Animals were housed in the Animal Resource Center (Faculty of Medicine) in accordance with Canadian Council on Animal Care Guidelines and with the approval of the Animal Care Committee of the Faculty of Medicine. TISSUE PREPARATION All animals were sacrificed by Euthanyl overdose (MTC Pharmaceuticals, Cambridge, Ontario) intravenously through the lateral ear vein. The medial and lateral menisci were carefully removed aseptically from the knee joint to avoid capsular contamination of the tissue. The quality of Fig. 1. Gross morphological assessment of the medial and lateral menisci of non-operated and ACL transected animals at 3 and 8 weeks. (A) Gross morphological appearance of the medial and lateral menisci of nonoperated and ACL transected animals at 3 and 8 weeks. Note the prominant bucket-handle tear in the posterior aspect of the medial meniscus at 8 weeks post-acl transection and the absence of tears in the lateral menisci at either of the times after surgery. (B) Scoring of the gross morphological changes was assessed as described in the Material and Methods section. Changes over time in the surface of menisci after ACL transection were assessed by India ink staining. the meniscal surfaces was assessed by the India ink method of Meachim 24 and graded as described by Adams et al. 12 as follows: grade 1=normal; grade 2=minimal fibrillation; grade 3=moderate surface fibrillation but no tears; grade 4=severe fibrillation, incomplete tears; grade 5=complete tears; bucket-handle tears or multiple incomplete tears. For water content, the fresh wet weight was immediately determined, followed by lyophilization to constant weight, and dry weight determination. These values were used to define the percentage of water content of the tissue samples. Samples were then stored at 80 C prior to processing. HISTOLOGY AND IMMUNOHISTOCHEMISTRY After sacrifice, the medial and lateral menisci were resected and immediately fixed in 10% formalin buffer (ph 7.4) containing 0.5% of cetylpyridinium chloride, dehydrated through graded alcohols, cleared with xylene, and embedded in paraffin. Transversal sections of the whole menisci were cut at 6 μm and mounted on aminoalkylsilane-treated slides. The sections were then stained with hematoxylin and eosin for general morphology or safranin-o/fast green for proteoglycan staining. Sections used for the figures were consistently at approximately 300 μm from the tibial face of the menisci. Immunohistochemistry was performed with antibodies raised against collagen types I, II and III (Medicorp, Montreal, Quebec). The immunohistochemical localization

3 58 M.-P. Hellio Le Graverand et al.: Structural changes in menisci in experimental osteoarthritis Table I Water content in medial and lateral menisci Medial (N=6) Control % Water content S.D. P-value (BonFerroni post-test) Lateral (N=6) 3 weeks post-acl-t 8 weeks post-acl-t (a***) (b*) Control weeks post-acl-t 8 weeks post-acl-t (a): 3 weeks post-acl-t vs. control; (b): 8 weeks post-acl-t vs control; *P<0.05; **P<0.01; ***P< Fig. 2. Structural findings of the medial and lateral menisci at 8 weeks post-acl transection. (A) Schematic representation of the medial meniscus from normal, 3 weeks and 8 weeks post-acl transected rabbit knees. The photographs displayed in (B) were taken from sections of the medial meniscus stained with Safranin-O/fast green and correspond to the indicated zones (zones 1 9). (B) Histology of the medial meniscus from normal, 3 weeks and 8 weeks post-acl transected rabbit knees. Zone 5 represents the region where intrameniscal fissures form, characterized by the absence of staining for PG and absence of cells. Zone 6 represents the outer region of the zone of formation of intrameniscal fissure and stains strongly for PG the outermost connective tissue has been replaced by a new angiogenic and highly cellular connective tissue. Zone 8 corresponds to the outer side of the bucket-handle tear and exhibits large and abundant cell clusters staining strongly for PG. Original magnification, 10. techniques for collagen types I, II and III have been described previously.25 Briefly, serial sections were deparaffinized by two incubations in xylene for 5 min, and endogenous peroxydase activity was quenched by incubation in 0.3% hydrogen peroxide in distilled water for 30 min. Antigen retrieval was performed by incubating the section in trypsin (0.25%, Sigma) at 37 C for 30 min followed by hyaluronidase (1.45 IU/ml; Seikagaku corporation, Tokyo) and chondroitinase ABC (0.25 IU/ml; Seikagaku corporation, Tokyo) treatment at 37 C for 30 min. All subsequent incubations were carried out in the presence of 10% sheep serum to reduce non-specific

4 Osteoarthritis and Cartilage Vol. 9 No Fig. 4. Two types of cell clusters in the medial meniscus at 8 weeks post-acl transection. Original magnification, 40. Note the difference in proteoglycan staining for two types of cell clusters located on the outer edge of the bucket-handle tear. STATISTICAL ANALYSIS Fig. 3. Development of intrameniscal tears in the medial meniscus following ACL transection. Note the appearance of a fissure (B) along a zone of extracelullar matrix lacking PG staining and depleted of meniscal cells (A). Original magnification, 10. Water content values are given as mean±standard deviation of six rabbits. Comparisons were made using one-way ANOVA and the BonFerroni test was subsequently applied as a post-test when comparing pairs of group means. Differences with P-values of less than 0.05 were considered significant. Results binding. Sections were incubated with the primary antibody (mouse anti-human anti-type I collagen, 1/100 dilution or anti-type II and anti-type III collagen, 1/5000 dilution, Medicorp, Montreal, Quebec) overnight at 4 C. Amplification of the primary antibody reaction was performed by incubating sections with peroxidase-conjugated sheep antimouse secondary antibody (Roche Diagnostics, Laval, Quebec) for 30 min at room temperature. The control studies were performed by substitution of the primary antibody with Tris buffered saline (TBS). Binding was visualized by incubation of sections using a diaminobenzidine substrate kit (Roche Diagnostics, Laval, Quebec). The slides were counterstained with hematoxylin. The results of immunostaining were analyzed qualitatively in a blinded fashion by an experienced observer. GROSS PATHOLOGY AND GRADING OF THE MENISCI At necropsy, all specimens from the ACL transection groups exhibited complete transection of the ACL. Both medial and lateral menisci from operated joints were altered, in particular the posterior region of the medial meniscus. The medial meniscal lesions were more severe with time after surgery and appeared earlier than in the lateral meniscus. The grading of both menisci after India ink staining is presented in Fig. 1. At 3 weeks post-acl transection, 7/12 of the medial menisci presented with incomplete vertical posterior tears, while only 1/12 of those medial menisci presented with a bucket-handle tear. At 8 weeks post-acl transection, all of the medial menisci had severe bucket-handle tears (Fig. 1, Panel A). In contrast, only superficial changes were observed in the lateral

5 60 M.-P. Hellio Le Graverand et al.: Structural changes in menisci in experimental osteoarthritis Fig. 5. Immunostaining of the medial meniscus. Staining of normal, 3 weeks and 8 weeks post-acl transection medial meniscus with a 1(I) chain-specific monoclonal antibody. Original magnification, 10. meniscus. No tears were observed at either 3 and 8 weeks post-acl transection. Menisci from the non-operated controls were all intact. Only a small volume (<0.1 ml) of synovial fluid was present in control joints from non-operated rabbits. In contrast, the volume of synovial fluid was increased to 2 ml in joints from 3 weeks post-acl transection rabbits. At 8 weeks post-acl transection, the volume of synovial fluid was less abundant than at 3 weeks, but still elevated to 0.5 ml. The synovial fluid was less viscous than normal and had a yellowish color in the operated joints. Gross characteristics of OA such as large osteophyte formation were observed but no overt fibrillation of articular surfaces was detected at the 3 and 8 weeks time points. MENISCAL WATER CONTENT The percent water content of the menisci are presented in Table I. The water content of the menisci of unoperated joints averaged 62.27%±1.2 (S.D.) in the medial menisci and 63.66%±2.15 in the lateral menisci. The mean values for water content of the lateral menisci were unchanged after ACL transection. In contrast, the mean values for water content of the medial menisci were significantly elevated after 3 and 8 weeks post-acl transection (P<0.001 and P<0.05, respectively). HISTOPATHOLOGY Compared to normal medial menisci, at 3 weeks postacl transection the inner part of the medial meniscus stained less intensely for PG than collagen (Fig. 2, zone 4). Three weeks after surgery, fissures within the midsubstance of the medial menisci starting from the middle region and extending toward the posterior region were observed, with characteristic depletion of cells in either both or the upper or the lower zones of the fissures and absence of proteoglycan staining [Fig. 2, zone 5 and Fig. 3(A), (B)]. The outer part of the meniscus substance exhibited an increased cell number, as well as formation of small clusters along the outer edge of the fissures and abnormal organization of the collagen and increased proteoglycan staining [Fig. 2, zone 6 and Fig. 3(A), (B)]. The normal connective tissue forming the outer ridge was replaced by a highly vascularized and cellular connective tissue rich in PG (Fig. 2, zones 3 and 6). At 8 weeks post-surgery, the extracellular matrix of the medial meniscus showed marked degenerative changes: bucket-handle tears, extensive alteration in the pattern of proteoglycan staining with areas of high intensity and areas of absence of staining, extreme alteration in cell distribution with acellular areas and areas of large and abundant cell clusters (Fig. 2, zones 7, 8 and 9; Fig. 4).

6 Osteoarthritis and Cartilage Vol. 9 No Fig. 6. Immunostaining of the medial meniscus. Staining of normal, 3 weeks and 8 weeks post-acl transection medial meniscus with a α1(ii) chain-specific monoclonal antibody. Original magnification, 10. The histological appearance of the lateral meniscus was not dramatically altered, showing only rare surface fibrillations but no remarkable extracellular matrix or cell modification. IMMUNOHISTOCHEMISTRY To determine whether the observed histologic changes for the collagens were reflected by changes in the distribution of collagen, the content and distribution of types I, II and III collagen both medial and lateral menisci at 3 and 8 weeks post-acl transection were analyzed using immunohistochemical techniques. The distribution of type I, II and III collagen staining was uniform thoughout both normal medial and lateral meniscal tissue (Figs 5, 6, 7 and 8). At 3 weeks post-acl transection, in the medial meniscus, types I, II and III collagen staining intensity increased in the outer region adjacent to the fissure which was associated with the increased proteoglycan staining region observed following histological Safranin-O/fast-Green staining (Figs 5, 6, 7). In addition, type II collagen staining was increased in the inner part of the medial meniscus (Fig. 6). This altered pattern of staining for type I, II and III collagens in the medial meniscus remained very evident at 8 weeks post- ACL transection (Figs 5, 6, 7). At 3 and 8 weeks post-acl transection, in the lateral meniscus type I and III collagen staining was increased throughout the tissue (Fig. 8). In contrast, type II collagen staining was not overtly altered in the lateral meniscus following ACL transection. Discussion The results presented in this report demonstrate that transection of the ACL can lead to structural changes in rabbit knee menisci, and that the observed changes are both meniscus-specific and time-dependent. The present study confirms and significantly extends the previous findings observed by Adams et al. in the canine model of OA following ACL transection. 12 These authors described gross pathological changes in menisci, in particular in the medial meniscus. 12 In the present study, none of the lateral menisci presented with severe lesions while all of the medial menisci had grade 5 changes at 8 weeks post-acl transection and 70% had grade 4 or 5 at 3 weeks post-acl transection. This compares with the lateral menisci where 50% had grade 1, 38% had grade 2 and 12% had grade 3 at 8 weeks post-acl transection. The findings that the medial meniscus was more severely altered than the lateral meniscus after ACL transection is in agreement with a number of clinical studies. In a recent review of the literature, Bellabarba et al. reported that the importance of meniscal pathology associated with ACL insufficiency stems from the increased incidence of meniscal tears with chronic instability of the knee joint. 26

7 62 M.-P. Hellio Le Graverand et al.: Structural changes in menisci in experimental osteoarthritis Fig. 7. Immunostaining of the medial meniscus. Staining of normal, 3 weeks and 8 weeks post-acl transection medial meniscus with a 1(III) chain-specific monoclonal antibody. Original magnification, 10. There is some controversy regarding whether the medial or lateral meniscus is most often injured. However, in the latter review, while a predominance of lateral meniscal tears with acute ACL rupture was reported, the incidence of medial meniscal tears increased significantly with chronic ACL insufficiency.26 In the present study, the time periods following surgery (i.e. 3 and 8 weeks post-acl transection) are consistent with a chronic joint instability condition. In addition, in a recent retrospective study of 1740 knee joints examined arthroscopically, with the objective of determining possible correlations between meniscal and cartilage lesions, the majority of the abnormal menisci were located in the medial compartment.22 Meniscal tears were four times more common in the medial compartment when compared with their frequency in the lateral compartment.22 Moreover, longitudinal, bucket-handle and complex tears of the medial meniscus were significantly associated more with articular cartilage damage than horizontal cleavage, flap or radial tears.22 Interestingly, medial meniscal lesions were more frequently associated with femoral and tibial cartilage degeneration than were lateral meniscal injuries (P<0.001).22 Nevertheless, in the present study, the observed gross morphological changes suggest that early alterations in the material properties of the meniscal tissue may occur and these could lead to abnormal meniscal function. Any alterations in the major functions of load bearing, load distribution and shock absorption of the menisci could lead to disturbance in the underlying articular cartilage tissues, leading to cartilage degeneration. The preponderance of medial meniscal alterations in the present study is consistent with observation in patients. The present study describes for the first time dramatic histologic alterations of the meniscal tissue following ACL transection. The medial meniscus-specific histologic findings observed at 3 weeks post-acl transection including the remodeling of the extracellular matrix (i.e. alteration of the matrix PG staining and collagen organization) and cell depletion, correlated with the appearance of the posterior tears. As sections were cut from the bottom to the top of the menisci, fissures within the mid-substance of the medial menisci were observed reproducibly. The location of these fissures suggests they are the point of origin of the subsequent longitudinal posterior or bucket-handle tears. The location of the intra-meniscal tears observed in the rabbit model coincide with the MRI images of Grade II and III menisci reported by Zuelzer et al.27 The areas of cell loss along the outer side of the fissure (3 weeks post-acl transection) and areas of large cell clusters along the bucket-handle tears (8 weeks post-acl transection) in the medial meniscus is also similar to the classic observation of chondrocyte clusters in proximity to fissured articular cartilage of osteoarthritic joints.28,29 Extracellular matrix staining of collagen in this model of OA was investigated and revealed significant increases in extracellular matrix staining for collagens type I, II and III in the medial meniscus and type I and III in the lateral

8 Osteoarthritis and Cartilage Vol. 9 No Fig. 8. Immunostaining of the lateral meniscus. Staining of normal, 3 weeks and 8 weeks post-acl transection lateral meniscus with 1(I) and 1(III) chain-specific antibodies. Original magnification, 10. meniscus. This suggests increased collagen deposition. However, as collagen fiber separation and later loss of collagen fiber orientation have been reported in dog menisci,30 it cannot be excluded that fiber disorganization leads to greater antibody accessibility and therefore more staining. Cautious interpretation of safranine-o staining must also be undertaken because of the potential loss of GAG during tissue processing. However, cetylpyridinium chloride was incorporated into the tissue processing to minimize non-specific losses. In addition, increased staining could occur due to increased epitope exposure, particularly in the pathological specimens that have higher water content (i.e. medial meniscus) and apparently looser fibrillar meshwork. However, the increased staining of the lateral meniscus following ACL transection for types I and III collagen, in the absence of increased water content, may argue against this explanation for the findings. As the analysis of the meniscal water content revealed no variations in water content in the lateral meniscus, the significant increase in water content in the medial meniscus, the observed tears, as well as the biochemical changes suggest a more profound disruption of the collagen network in the medial meniscus. The increase in water content could also be related to the increase in proteoglycan content as evidenced by areas of intense safranine-o staining. In conclusion, the present study demonstrates that reproducible structural and biochemical changes occur in menisci prior to cartilage changes following ACL transection in this model. The appearance of significant medial meniscal changes prior to cartilage changes suggests that the medial meniscus is one of the first tissues overtly involved in the development of OA following ACL transection. The unique increase in type II collagen deposition within the medial meniscus may represent an adaptation to the increased stress on the meniscus caused by joint instability. Future studies will determine to what extent the structural and biomechanical changes in the menisci following ACL transection could contribute to the subsequent degeneration of the underlying cartilage leading to OA. Acknowledgments The authors acknowledge the support of the Medical Research Council of Canada, The Arthritis Society and La Socie te Franc aise de Rhumatologie for these studies. MPHLG is supported by a Fellowship from The Arthritis Society and DAH is the Calgary Foundation-Grace Glaum Professor in Arthritis Research. The authors thank Dr Cyril B. Frank (Division of Orthopaedic Surgery, University of Calgary) for his critical review of the manuscript, Leona

9 64 M.-P. Hellio Le Graverand et al.: Structural changes in menisci in experimental osteoarthritis Barclay for excellent technical assistance in histological aspects of the study, and Paul Sciore, Carol Reno and Ray Boykiw for assistance in some molecular aspects of the study. References 1. Pond MJ, Nuki G. Experimentally-induced osteoarthritis in the dog. Ann Rheum Dis 1973;32: McDevitt C, Gilbertson E, Muir H. An experimental model of osteoarthritis; early morphological and biochemical changes. J Bone Joint Surg [Br] 1977; 59: McDevitt CA, Muir H. Biochemical changes in the cartilage of the knee in experimental and natural osteoarthritis in the dog. J Bone Joint Surg [Br] 1976;58: Vignon E, Arlot M, Hartmann D, Moyen B, Ville G. Hypertrophic repair of articular cartilage in experimental osteoarthrosis. Ann Rheum Dis 1983;42: Brandt KD, Braunstein EM, Visco DM, O Connor B, Heck D, Albrecht M. 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Lab Invest 1985;53: Stockwell RA, Billingham ME. Early response of cartilage to abnormal factors as seen in the meniscus of the dog knee after cruciate ligament section. Acta Biol Hung 1984;35: Abbreviations ACL, anterior cruciate ligament; COL1, type I collagen; COL2, type II collagen; COL3, type III collagen; GAG, glycosaminoglycan; OA, osteoarthritis; PG, proteoglycan; TBS, Tris-buffered saline